Conference Agenda

Physical forces are critical for the development, maintenance, and adaptation of various tissues in the body including bone. Bone adapts to mechanical loads through actions of bone cells: osteoclasts that specialize in bone resorption; osteoblasts that produce bone; and osteocytes embedded in bone matrix that sense mechanical forces and coordinate bone adaptation. ATP is a ubiquitous intracellular molecule critical for cellular bioenergetics, which is also one of the first signals released from bone cells that experience mechanical stimulation. Extracellular ATP is degraded by ecto-ATPases to form ADP and eventually adenosine. We developed a theoretical model describing mechanically induced ATP and ADP release, followed by their extracellular diffusion and degradation. The model was validated using experimental data for calcium signaling, ATP and vesicular release upon mechanical stimulation of a single osteoblast, and then scaled to a tissue-level injury. The route and amount of ATP release depended on mechanical forces, ranging from vesicular release of small ATP boluses upon membrane deformation, to leakage of ATP through resealable plasma membrane tears, to spillage of cellular content due to destructive forces. The total amount of ATP released indicated the degree of injury and determined the maximal distance from the injury at which responses were stimulated. The peak ATP concentration discriminated between injuries that released similar amounts of ATP due to differences in cell repair, and determined signal propagation velocity. ADP-mediated signaling was relevant for larger tissue-level injuries, conveying information about the proximity to the injury site and its geometry. After their release from an injury site, ATP, ADP, and adenosine signal through purinergic receptors, including seven P2X ATP-gated cation channels, seven G-protein coupled P2Y receptors responsive to ATP and ADP, and four P1 receptors stimulated by adenosine. All bone cells express multiple P2 receptors. We used published studies of overexpressed individual P2 receptors to fit their dependencies on ATP concentration using the Hill equation, and experimentally examined the concentration dependences of P2-mediated calcium responses to ATP and ADP in osteoblasts/osteocytes and osteoclasts. Dependence of the calcium response on ATP concentration exhibited a complex pattern that was not explained by the simple addition of individual receptor responses. To deconvolute the contribution of individual receptors to the ATP dose-response curve, we constructed a mathematical model that combined the actions of the P2Y2 receptor that has the highest affinity to ATP and induces inositol trisphosphate (IP3) mediated calcium release, and the P2X7 receptor that has the lowest ATP affinity and allows calcium influx through the pores. The model predicted interactions between these P2 receptors at the levels of receptor interactions and cellular signaling, which were validated using CRISPR/Cas9-generated P2Y2 and P2Y7 knockout cells. Our studies demonstrate how spatiotemporal signals provided by extracellular ATP and ADP encode the information regarding severity, scale and proximity of the mechanical stimulus, allowing for a well-choreographed tissue responses to physical forces.